A great diversity of oligosaccharide structures and many types of glycopeptides are found in nature, and these are synthesized, in part, by a large number of glycosyltransferases. Glycosyltransferases catalyze the synthesis of glycolipids, glycopeptides and polysaccharides by transferring an activated mono- or oligosaccharide residue from a donor to an existing acceptor molecule to initiate or elongate a carbohydrate chain. A catalytic reaction is believed to involve the recognition of both the donor and acceptor by suitable domains of the glycosyltransferase, as well as the catalytic site of the enzyme.
More than 30% of all therapeutic proteins and many potential peptide therapeutics are glycosylated peptides. It is well known in the art that the attachment of the correct glycan structure can play a key role in the folding, biological activity, biodistribution and pharmacological efficacy of therapeutic peptides. Furthermore, glycosylation is a critically important factor influencing the in vivo half life and immunogenicity of therapeutic peptides. Indeed, humans will typically tolerate only those biotherapeutics that have particular types of carbohydrate attachments and will often reject glycoproteins that include non-mammalian oligosaccharide attachments. For instance, poorly glycosylated peptides are recognized by the liver as being “old” and thus, are more quickly eliminated from the body than are properly glycosylated peptides. In contrast, hyperglycosylated peptides or incorrectly glycosylated peptides can be immunogenic.
The production of a recombinant glycopeptide, in contrast to a recombinant non-glycosylated peptide, requires that a recombinantly produced peptide is subjected to additional processing steps, either in vivo within the cell or in vitro after the peptide has been produced by the cell. The peptide can be treated enzymatically, using a glycosyltransferase to introduce one or more glycosyl groups onto the peptide by covalently attaching the glycosyl group or groups to the peptide.
The production of a glycopeptide by external in vitro-steps of peptide processing can be time consuming and costly. This is due, in part, to the burden and cost of producing recombinant glycosyltransferases for the in vitro glycosylation of peptides and glycopeptides to produce glycopeptide therapeutics. As the demand and usage of recombinant glycotherapeutics increases, new methods are required in order to prepare glycopeptides more efficiently.
Moreover, as more and more glycopeptides are discovered to be useful for the treatment of a variety of diseases, there is a need for methods that lower the cost of their production. Further, there is also a need in the art to develop methods of more efficiently producing recombinant glycopeptides for use in developing and improving glycopeptide therapeutics.
Glycosyltransferases and their use for the glycosylation of proteins are disclosed in WO 2003/031464 A2.
Sialyltransferases constitute a family of glycosyltransferases that catalyze the posttranslational transfer of sialic acid (N-acetylneuraminic acid) to acceptor oligosaccharide substrates at terminal positions on glycoproteins and glycolipids (Paulson et al., 1989, J. Biol. Chem. 264: 17615-17618). It is estimated that the human genome encodes more than 20 different sialyltransferases required to synthesize all known sialo-oligosaccharide structures present in mammalian cells, but only 16 distinct human sialyltransferase cDNAs have been cloned (Tsuji S et al., 1996, Glycobiology 6: 5-7; Tsuji S, 1996, J. Biochem. 120:1-13; Weinstein J et al., 1982, J. Biol. Chem. 257: 13835-13844). Originally, sialyltransferases were biochemically purified and their cDNAs were cloned using N-terminal sequences. Comparison of the obtained cDNA sequences revealed two highly conserved regions, termed the L- and S-sialylmotifs, that participate in substrate binding. Subsequently, several sialyltransferases were cloned by PCR using degenerate primers designed within the sialylmotifs or by expression cloning (Nara K et al., 1994, Proc. Natl. Acad. Sci. USA 91: 7952-7956; Nakayama J et al., 1996, J. Biol. Chem. 271: 3684-3691; Nakayama J et al., 1995, Proc. Natl. Acad. Sci. USA 92: 7031-7035). Gene cloning by differential display adds an entirely different approach to the identification of novel sialyltransferases with putative functional significance in disease-related processes.
Sialyltransferases differ in their substrate specificity and tissue distribution, and they are classified into four families according to the carbohydrate linkages they synthesize: the ST3Gal-, ST6Gal-, ST6GalNAc-, and ST8Sia-families. The members of each family exhibit strong activity towards certain acceptor groups, although the substrate specificities of these enzymes overlap; one linkage can be synthesized by multiple enzymes.
One such particular sialyltransferase that has utility in the development and production of therapeutic glycopeptides is N-acetylgalactosamine-α2,6-sialyltransferase (ST6GalNAcI) which catalyzes the transfer of sialic acid from a sialic acid donor to a sialic acid acceptor. Full length chicken ST6GalNAcl enzyme, for example, is disclosed by Kurosawa et al. (1994, J. Biol. Chem., 269:1402-1409).
In the past, there have been efforts to increase the availability of recombinant sialyltransferases for the in vitro production of glycopeptides.
EP 0 737 745 A1 and U.S. Pat. No. 5,032,519 of the Institute of Physical & Chemical Research refer to the use of E. coli for producing a secreted version of a protein comprising a portion, i.e. active domain, that is derived from ST6GalNAcI and is responsible for its activity.
WO 2007/056524 A2 of Neose Technologies Inc. describes methods of producing a modified ST6GalNAcI polypeptide, the method comprising growing a recombinant prokaryotic host cell under conditions suitable for expression of the modified ST6GalNAcI polypeptide in prokaryotic host cells. These modified ST6GalNAcI polypeptides are chimeric polypeptides comprising a first portion from a Gal-β1,3GalNAc-α2,3-sialyltransferase (ST3GalI) polypeptide and a second portion from a GalNAc-α-2,6-sialyltransferase I (ST6GalNAcI) polypeptide. Modified ST6GalNAcI polypeptides can further be truncated polypeptides lacking all or portion of the ST6GalNAcI signal domain, all or a portion of the ST6GalNAcI transmembrane domain, and/or all or a portion of the ST6GalNAcI stem domain in eukaryotic or prokaryotic host cells.
US 2006/0234345 A1 of Neose Technologies Inc. discloses a method of producing a soluble eukaryotic glycosyltransferase in a prokaryotic microorganism that has an oxidizing environment, by a) expressing a nucleic acid that encodes the eukaryotic glycosyltransferase in the prokaryotic microorganism; and then b) growing the prokaryotic microorganism under conditions that allow expression of the soluble active eukaryotic glycosyltransferase within a cellular compartment of the prokaryotic microorganism.
Skretas et al. (2009, Microbial Cell Factories, 8:50) relate to a method for the expression of the human sialyltransferase ST6GalNAcI in engineered E. coli strains which possess certain types of oxidative cytoplasm or which co-express the molecular chaperones/co-chaperones trigger factor, DnaK/DnaJ, GroEL/GroES, and Skp, and can produce greatly enhanced amounts of soluble ST6GalNAcI.
However, the capacity of E. coli for protein folding and forming disulfide bonds is not sufficient although there are a number of tools developed to overcome these limitations. Furthermore, the high expression yield of recombinant proteins in E. coli expression systems can often lead to the accumulation of aggregated, insoluble proteins that form inclusion bodies which can be a significant hindrance in obtaining soluble, active proteins (Brondyk W. H., 2009, Methods in Enzymology, Vol. 463, Ch. 11).
To overcome the problems associated with recombinant sialyltransferase production in E. coli cultures, insect cell culture systems have been developed.
US 2006/0246544 A1 and US 2008/0207487 A1 disclose a method of making a composition that includes a recombinant polypeptide, e.g. sialyltransferases, wherein the polypeptide is expressed in an insect cell (e.g., using a baculoviral expression system) and wherein the composition is essentially free of endoglycanase activity. The method includes subjecting a mixture including the polypeptide to mixed-mode chromatography including the steps of: (i) contacting the mixture and a mixed-mode chromatography medium; and (ii) eluting the polypeptide from the mixed-mode chromatography medium generating a flow-through fraction comprising the polypeptide.
However, the complexity of the baculovirus-insect cell expression system, the limited storage stability of the required viral seed stocks and the requirement of very high virus titers for an efficient infection can limit its use for large-scale bioproduction. Furthermore, viral vectors such as baculovirus have been shown to be able to infect mammalian cells, particularly human cells (Boyce F M and Buchner N L, 1996, Proc. Natl. Acad. Sci. USA 93:2348-2352; Lundstrom et al., 2001, Cytotechnology, 35: 213-221). Thus, these vectors pose a threat concerning safety issues, especially when applied for large-scale recombinant protein production, where large volumes of infected cells are handled.
An alternative to overcome the described limitations of the use of insect cell culture systems is the use of mammalian cell systems for the manufacture of recombinant sialyltransferases.
WO 2005/121332 A2 of Neose Technologies Inc. discloses methods of producing isolated truncated ST6GalNAcI polypeptide that lacks all or a portion of the ST6GalNAcI signal domain, all or a portion of the ST6GalNAcI transmembrane domain, and/or all or a portion of the ST6GalNAcI stem domain in prokaryotic and insect host cells and generally mentions that the polypeptide may also be produced in mammalian cells.
U.S. Pat. No. 5,032,519 of the University of California describes methods of transfecting a host cell, e.g. a CHO cell, with a vector carrying a gene which expresses a glycosyltransferase that has the membrane anchor and most of the stem region replaced with a cleavable secretion signal segment. The resulting soluble glycosyltransferase, when expressed in the cell, is secreted by the cell. The secreted soluble glycosyltransferase is then separated from the cell culture media for use in industrial applications or carbohydrate synthesis research. Further, U.S. Pat. No. 5,032,519 discloses a method of purifying a soluble glycosyltransferase by using an affinity chromatography.
However, none of the mentioned documents relating to the production of recombinant sialyltransferases in mammalian cells discloses a method for providing a recombinant sialyltransferase which is highly active, purified to a pharmaceutical grade and amenable to large scale production.
Therefore, a need still exists for efficient methods of production of recombinant sialyltransferases having activity and purity that are suitable for “pharmaceutical-scale” processes and reactions, especially for the production of glycopeptide therapeutics. Thus, the problem underlying the present invention is to provide such methods for producing recombinant sialyltransferases.